235
Furukawa and Ishii 1967 ; Hama 1969 ; Fay 1974 ; Zotterman 1943 ). Noise signals at
frequencies other than the reference frequency are rejected by the lock-in amplifi er
and do not affect the measurements.
Threshold tuning curves were constructed by characterizing the input–output
measurements of the RMS amplitudes of the evoked saccular potentials over the
range of stimulus intensities at the tested frequencies. Background noise measurements were also recorded for 8–10 repetitions of the stimulus interval at each of the
test frequencies with no auditory stimulus present prior to the recording of each
threshold tuning curve, and were then used to establish subthreshold saccular potential response levels. Auditory threshold at each stimulus frequency was designated
as the lowest stimulus intensity that evoked a saccular potential that was at least 2
SD above the background noise measurement. The frequency that evoked the lowest
saccular potential threshold was defi ned as the best frequency.
2.3 Auditory Evoked Potential (AEP) Recordings
AEP tuning curves were determined from 7 Hawaiian sergeant fi sh collected in late
July (6 males, 1 female; SL = 132.4 ± 7.0 SD mm; BM = 100.1 ± 18.5 SD g). To
ensure fi sh were in similar reproductive condition to those used for saccular potential recordings, these fi sh were collected and tested immediately prior to the fi sh that
were collected and shipped to the University of Washington in early August. AEPs
were performed identical to that described in Maruska et al. ( 2007 ), except that
additional stimulus frequencies in 25 Hz increments were tested between 100 and
400 Hz. This fi ner frequency resolution was performed to more closely match the
frequencies used in saccular potential recordings, and because natural A. abdominalis sounds and best hearing sensitivity is within this low frequency spectral range.
Briefl y, immobilized fi sh were positioned in an experimental tank (30 cm diameter,
36.5 cm high, water level 29.5 cm high; fi sh positioned 16.5 cm above speaker)
above an underwater speaker (UW-30, Lubell Labs) and stainless steel sub-dermal
electrodes (Rochester Electro-Medical, Inc.; 6–12 kΩ) were placed beneath the skin
in the head musculature above the hindbrain (recording electrode) and between the
eyes (reference electrode). Fish were continuously ventilated with fresh seawater
during all experiments. Acoustic stimuli were generated with a Cambridge
Electronics Design (CED, Cambridge, UK) Micro 1401 controlled by Spike 2 software and delivered to the speaker via CED 3505 attenuator and amplifi er (UMA
352, Peavey Electronics). Stimuli consisted of 2000 repetitions of 20 ms pulses
(for ≥200 Hz: 10 ms plateau with rise and fall times of 5 ms; for 100 Hz: 10 ms
plateau, rise, and fall; for 80 Hz: 13 ms plateau, rise, and fall). Sequential alternation
of stimulus phase during the 2000 repetitions was used to eliminate stimulus artifacts in the AEP recordings. Trials began at suprathreshold intensities and were
decreased in 5 dB steps to a sound level below the presumed threshold before
moving to the next test frequency. Sound levels produced by the speaker were calibrated with a B&K hydrophone (model 8103; sensitivity −211 dB re: 1 V/μPa)
Comparison of Electrophysiological Auditory Measures in Fishes
Furukawa and Ishii 1967 ; Hama 1969 ; Fay 1974 ; Zotterman 1943 ). Noise signals at
frequencies other than the reference frequency are rejected by the lock-in amplifi er
and do not affect the measurements.
Threshold tuning curves were constructed by characterizing the input–output
measurements of the RMS amplitudes of the evoked saccular potentials over the
range of stimulus intensities at the tested frequencies. Background noise measurements were also recorded for 8–10 repetitions of the stimulus interval at each of the
test frequencies with no auditory stimulus present prior to the recording of each
threshold tuning curve, and were then used to establish subthreshold saccular potential response levels. Auditory threshold at each stimulus frequency was designated
as the lowest stimulus intensity that evoked a saccular potential that was at least 2
SD above the background noise measurement. The frequency that evoked the lowest
saccular potential threshold was defi ned as the best frequency.
2.3 Auditory Evoked Potential (AEP) Recordings
AEP tuning curves were determined from 7 Hawaiian sergeant fi sh collected in late
July (6 males, 1 female; SL = 132.4 ± 7.0 SD mm; BM = 100.1 ± 18.5 SD g). To
ensure fi sh were in similar reproductive condition to those used for saccular potential recordings, these fi sh were collected and tested immediately prior to the fi sh that
were collected and shipped to the University of Washington in early August. AEPs
were performed identical to that described in Maruska et al. ( 2007 ), except that
additional stimulus frequencies in 25 Hz increments were tested between 100 and
400 Hz. This fi ner frequency resolution was performed to more closely match the
frequencies used in saccular potential recordings, and because natural A. abdominalis sounds and best hearing sensitivity is within this low frequency spectral range.
Briefl y, immobilized fi sh were positioned in an experimental tank (30 cm diameter,
36.5 cm high, water level 29.5 cm high; fi sh positioned 16.5 cm above speaker)
above an underwater speaker (UW-30, Lubell Labs) and stainless steel sub-dermal
electrodes (Rochester Electro-Medical, Inc.; 6–12 kΩ) were placed beneath the skin
in the head musculature above the hindbrain (recording electrode) and between the
eyes (reference electrode). Fish were continuously ventilated with fresh seawater
during all experiments. Acoustic stimuli were generated with a Cambridge
Electronics Design (CED, Cambridge, UK) Micro 1401 controlled by Spike 2 software and delivered to the speaker via CED 3505 attenuator and amplifi er (UMA
352, Peavey Electronics). Stimuli consisted of 2000 repetitions of 20 ms pulses
(for ≥200 Hz: 10 ms plateau with rise and fall times of 5 ms; for 100 Hz: 10 ms
plateau, rise, and fall; for 80 Hz: 13 ms plateau, rise, and fall). Sequential alternation
of stimulus phase during the 2000 repetitions was used to eliminate stimulus artifacts in the AEP recordings. Trials began at suprathreshold intensities and were
decreased in 5 dB steps to a sound level below the presumed threshold before
moving to the next test frequency. Sound levels produced by the speaker were calibrated with a B&K hydrophone (model 8103; sensitivity −211 dB re: 1 V/μPa)
Comparison of Electrophysiological Auditory Measures in Fishes